Platelet membrane camouflaged AIEgen‐mediated photodynamic therapy improves the effectiveness of anti‐PD‐L1 immunotherapy in large‐burden tumors

Abstract Although immunotherapy has achieved recent clinical success in antitumor therapy, it is less effective for solid tumors with large burdens. To overcome this challenge, herein, we report a new strategy based on platelet membrane‐camouflaged aggregation‐induced emission (AIE) luminogen (Plt‐M@P) combined with the anti‐programmed death ligand 1 (anti‐PD‐L1) for tumoral photodynamic‐immunotherapy. Plt‐M@P is prepared by using poly lactic‐co‐glycolic acid (PLGA)/PF3‐PPh3 complex as a nanocore, and then by co‐extrusion with platelet membranes. PF3‐PPh3 is an AIE‐active conjugated polyelectrolyte with photosensitizing capability for photodynamic therapy (PDT). Plt‐M@P exhibits superior tumor targeting capacity in vivo. When applied in small tumor‐bearing (~40 mm3) mice, Plt‐M@P‐mediated PDT significantly inhibits tumor growth. In tumor models with large burdens (~200 mm3), using Plt‐M@P‐mediated PDT or anti‐PD‐L1 alone is less effective, but the combination of both is effective in inhibiting tumor growth. Importantly, this combination therapy has good biocompatibility, as demonstrated by the absence of damage to the major organs, especially the reproductive system. In conclusion, we show that Plt‐M@P‐mediated PDT can improve anti‐PD‐L1 immunotherapy by enhancing antitumor effects, providing a promising strategy for the treatment of tumors with large burdens.


| INTRODUCTION
Immunotherapy has achieved accelerated development in particular for clinical cancer treatment over the past few years, among which chimeric antigen receptor-modified T (CAR-T) cell therapy and the inhibitors of immune checkpoints are the most well-known. [1][2][3] The axis of programmed cell death protein 1 (PD-1) and programmed death ligand 1 (PD-L1) plays a critical role in immune homeostasis and the application of immune checkpoint inhibitors to block PD-1/PD-L1 interaction shows recent clinical success. [4][5][6][7] Early clinical trials demonstrated that immunotherapies could achieve long-lasting remissions in patients with hematologic malignancies. [8][9][10] However, treatment of solid tumors with immunotherapies has yielded limited therapeutic benefits to date. [11][12][13] Although an active immunotherapy strategy is usually effective for small tumor burdens, it is much less so for large tumor burdens. [14][15][16] The key immunotherapies barriers within solid tumors, especially in large tumors, could be attributed to the immunosuppressive tumor microenvironment. 17 Preclinical data support the findings that large tumors are more immunosuppressive compared to small tumors. 16 The immunosuppressive state of the tumor microenvironment is enforced via immune cell subsets recruited or induced, such as T cell, dendritic cells, and natural killer cells. 18 It is suggested that targeting regulating immune cell populations may help convert the tumor microenvironment of large tumors to more closely resemble the immune infiltrate of small tumors. 16 Hence, strategies to remove these obstacles in large-burden tumors are primarily focused on the combination of different anticancer therapies, aiming to make the tumors more vulnerable to immunotherapy. [19][20][21] Photodynamic therapy (PDT) is a clinically used, minimally invasive therapeutic method. [22][23][24][25] The key element of PDT is the photosensitizer which is activated by irradiation with a specific wavelength of light. [26][27][28] Upon irradiation, photosensitizer generates highly cytotoxic reactive oxygen species (ROS) to cause oxidative stress-induced tumor cell death. 29,30 While traditional photosensitizers, such as porphyrin, phthalocyanine, and analogs, adopt large flat disc-like structures, are inclined to aggregate and experience strong intermolecular πÀπ stacking interactions. The excited states of such aggregates often decay to the ground state via nonradiative channels, suffering from aggregation caused quench (ACQ) effect which leads to the reduction of ROS generation efficiency. 31,32 In addition, both Ce6 and ICG are typical ACQ photosensitizers, and the higher the degree of their aggregation, the lower the yield of resulting ROS instead. 31,[33][34][35] The emergence of the photosensitizer with aggregation-induced emission (AIE) properties can overcome those challenges. While AIE photosensitizers stay in the aggregate state, the nonradiative decay processes are inhibited, and the excited state can thus be harvested for emission as well as ROS generation, exhibiting superior photostability and photosensitizing capacity. [36][37][38][39][40] Extensive evidences have been found, at the animal level, that AIE photosensitizers have good antitumor effects and opened up many opportunities with great potential for further employing PDT for tumor treatment in a clinical setting. [41][42][43][44] Hence, it is a pressing issue today to develop high-performance AIE photosensitizers to fulfill the need of the area. Recent studies reported that small tumors responded well to a single round of PDT but large tumors showed lower response to the same treatment. [45][46][47][48] This may be due to the limited penetration depth of light PDT and thus low inhibition efficiency for large-burden tumors. 48 In addition, the severe hypoxic state and more complex tumor microenvironment in large-burden tumors also result in discounted efficiency of PDT. 49 In addition to local tumor ablation, PDT can increase tumor immunogenicity by inducing tumor-associated antigens exposure, which improves the presentation of tumor-associated antigens and activation of T lymphocytes. [50][51][52] Furthermore, PDT enhances the expression of a number of pro-inflammatory cytokines, including interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-12 (IL-12), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ). 50,53 PDT has multiple effects on improving the intra-tumor microenvironment and has been demonstrated to improve the treatment outcome of immunotherapies on mice. [54][55][56][57][58][59] Marrache et al. encapsulated a photosensitizer, zinc phthalocyanine, with CpG to enhance pro-inflammatory cytokines release, dendritic cells maturation, and activation for metastatic breast cancer. 56 Our recent study also showed the combination of the efficient PDT with immunologic adjuvants Poly(I:C) for synergistic activation of the immune system for effective tumor treatment. 57 With these prior successes, we are intrigued to explore whether PDT can improve the immunosuppressive microenvironment and thus enhance the efficacy of immunotherapy against large-burden tumors.
The major adverse effects of PDT are photosensitivity reactions, including local skin redness, oozing, and hyperpigmentation. To reduce the occurrence of these adverse reactions, reducing the accumulation of photosensitizer at non-tumor sites is an effective means. 51,60 In recent years, nanoparticle (NP) drug carrier research has demonstrated to be exceptional to enhance PDT in the treatment of cancer via enhanced drug delivery mechanisms. 61 Cell membranes, including red blood cells, platelets, immune cells, stem cells, and cancer cells, have recently emerged as new sources of materials for drug delivery systems because of their enhanced biocompatibility, low immunogenicity, and active targeting abilities. 62,63 Among these materials, platelets have drawn research interest in drug delivery because of their capability to target specific sites and escape the immune system.
Platelet membrane also has the capacity to evade phagocytosis while in blood circulation. In addition, platelet membranes express P-selectin, a cell adhesion protein that can bind to CD44 receptors overexpressed in cancer cells, and thus, these platelet membrane-coated NPs showed greater uptake by tumor cells in vitro than plain NPs. 64 Similarly, platelet membrane-coated NPs exhibited a greater accumulation in tumor sites and exerted enhanced antitumor efficacy in vivo. 65 Recently, platelet membranes have also been used to coat NPs for enhanced cancer immunotherapy. 66,67 Based on these advantages of platelet membrane, platelet membrane-coated NPs hold promise to improve tumor microenvironment in large-burden tumors and make them more vulnerable to immunotherapy. Therefore, we envisioned that using platelet membrane-based drug delivery systems can improve the targeting of the AIE photosensitizer, so as to achieve desired therapeutic effects with minimal side-effects.
We demonstrate that Plt-M@P NPs are able to specifically target were purchased from Sigma-Aldrich and J&K Scientific Ltd. 1 H, 13 C, and 31 P nuclear magnetic resonance (NMR) spectra of small molecules, polymers, and polyelectrolytes were measured on a Bruker AV 500 spectrometer in deuterated solvents (chloroform or DMSO) using tetramethylsilane (TMS; δ = 0) as the internal reference. UV-vis absorption spectra were measured on a Shimadzu UV-2600 spectrophotometer. Photoluminescence (PL) spectra were recorded on a

| Preparation of Plt-M@P
Briefly, blood was collected from mice via the heart and placed in tubes containing EDTA (1.5 mg/ml) and stored at 4 C. The blood sample should be subjected to platelet extraction within 24 h. Equal volumes of PBS were used to dilute the blood samples. The volume ratio of diluted blood sample to platelet isolate was 1:1. Immediately afterward, the samples were centrifuged with a centrifugal force of 200g and a centrifugation time of 15 min. Note that the brakes cannot be applied after the sample centrifugation is completed. Carefully aspirate the platelet-rich plasma (top layer) from the sample and transfer to a new centrifuge tube. PBS was added to the plasma for washing, and after washing, it was centrifuged again (500g, 20 min). Platelets are obtained after two repeated washes. Platelet membranes are obtained by repeated freeze-thawing of platelets. 71 Briefly, platelets are placed in liquid nitrogen and frozen, then placed at room temperature to dissolve, and the operation is repeated five times. Platelet membranes were then obtained by high-speed centrifugation (4000g, 15 min).
The preparation of Plt-M@P is referred to the previous studies. 72

| Detection of ROS in solution
The equipment involved in the photodynamic system is shown in Figure S6. Light source, is a xenon lamp, purchased from Beijing zhonghuitingcheng Technology Co. Before conducting the photosensitivity test, the light source is fixed, and the light intensity close to the table is measured to a preset intensity (100 mW cm À2 ). Since the light intensity is checked before each test is conducted, it is possible to ensure the consistency of the light intensity. 9, 10-Anthracenediyl-bis (methylene)dimalonic acid (ABDA) is used as an indicator of ROS. A solution sample was prepared by mixing 50 μM of ABDA with the photosensitizers (P3-PPh3, PF3-PPh3, Ce6, and Plt-M@P) to be tested. The solution sample was added to a 3.5 cm Petri dish so that it could be well irradiated by white light. The samples were transferred to a cuvette for UV spectroscopy after a fixed time of light irradiation (30 s). It should be protected from light when performing UV spectroscopic measurements.
After the samples were tested, they were added to the petri dish again, the light was started, and so on in a cycle.

| Drug loading efficiency
For the preparation of Plt-M@P NPs, we put 2.0 mg of PF3-PPh 3 , and the Plt-M@P obtained was separated by ultrafiltration centrifugation, and the mass of PF3-PPh 3 in it was determined by UV spectroscopy to be 1.87 mg. Therefore, the loading efficiency of the drug can be deduced to be 93.5%.

| Cell culture
The 4T1 (Mouse breast cancer cell line) and HLF (Mouse lung fibroblasts) were purchased from the American Type Culture Collection.
The 4T1 cells were maintained in RPMI-1640 medium supplemented with 10% FBS, 100 U/ml penicillin, and streptomycin. The HLF cells were maintained in DMEM medium supplemented with 10% FBS, 100 U/ml penicillin, and streptomycin. All cells were incubated at 37 C with 5% CO 2 . The third and fourth passages were used for experiments.

| Blocking test
Briefly, 4T1 tumor cells were coincubated with CD44 antibody (20 ng/μl) for 12 h before incubation with Plt-M@P, and the intracellular fluorescence intensity was detected by CLSM after 24 h.

| Western blot
The proteins of PLGA/PF3-PPh 3 , Plt-M@P, and platelet membrane were extracted using RIPA buffer and then denatured at 100 C for 10 min. Samples (10 μl) were separated by 10% SDS-PAGE and then transferred to PVDF membranes. The membranes were blocked with 5% skimmed milk for 1 h at 37 C. Then, the membranes were incubated with the primary antibodies CD44 (1:1000 dilution), P-selectin (1:500 dilution), CD61 (1:1000 dilution), and CD41 (1:1000 dilution) overnight at 4 C. Next morning, the membranes were rewarmed at room temperature for 1 h and washed in TBST three times. HRPconjugated secondary antibody was added, and the membranes were incubated at 37 C for 1 h. The signals were detected by the enhanced ECL system with western blot exposure.    were euthanized and dissected, the tumor, spleen, liver, lung, heart, and kidney were obtained. Then the fluorescence signal in the organs was detected by the IVIS Spectrum imaging system (Ex = 500 nm, Em = 680 nm). Immediately afterward, the tissues were frozen sectioned and the accumulation of NPs in the tissues was observed by CLSM.

| TUNEL staining
The apoptotic of tumor cells was detected by using the One Step TUNEL Apoptosis Assay Kit. According to the manufacturer's instructions, the paraffin section tissue was incubated with terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) reaction mixture at 37 C for 1 h. Then the nuclei were stained with DAPI. Green fluorescein-labeled apoptotic cells were examined with CLSM (Zeiss).

| Flow cytometry
To detect the anti-tumor immune effects induced by PDT and anti-PD-L1, the peripheral blood of each group of mice was collected into ETDA-coated tubes. Then, 1 Â 10 5 cells were labeled with anti-CD4/FITC and CD8/APC antibodies for 20 min and washed two times with PBS. The cells were resuspended in 100 μl FACS buffer and then analyzed by flow cytometry (Beckman, CytoFLEX S).

| Immunofluorescence
Histological sections (4 μm thick) of organs and tumors were prepared. Deparaffinization was performed after the slides were

| Cytokine detection
The plasma levels of IL6 and IFN-α were measured using ELISA kits.
Plasma samples were extracted from the mice and diluted for analysis according to the ELISA Development Guide. All measurements were carried out in triplicate. Optical density (OD) was measured at a wavelength of 450 nm with a spectrophotometer (Molecular Devices).
Next, sections were stained with hematoxylin and eosin (H&E). Images were obtained through the microscope (Olympus).

| Ovarian follicle counting
Paraffin-embedded ovaries were sectioned to obtain 5-mm thick serial sections, and one of the four continuous ovarian sections was chosen to count the follicles. Follicles at different stages, including primordial follicles, primary follicles, secondary follicles, antral follicles, and atretic follicles, were determined as previously described. 74

| Statistical analysis
Data were presented as mean ± standard deviation (SD).  with AIE properties. 70 To further enhance the performance of this AIE photosensitizer system, PF3-PPh 3 was designed and successfully synthesized, and its synthesis path is shown in Figure 2a. Compound 1 was prepared using a previously reported method. 70 The neutral

| Pharmacokinetics and tumor targeting of Plt-M@P in vivo
The long circulation of NPs in vivo is not only the basis for the of NPs, such as P-selectin (receptor) and CD44 (ligand). 64,81,82 To confirm this hypothesis, immunofluorescence staining was used to estimate the expression level of CD44 in the heart, liver, spleen, lung and kidney (Figure 5g). Among them, there was almost no CD44 expression in the heart, liver, and kidney, while a high level of expression was found in the spleen and lung. CD44 expression in the tumor tissue was also at high levels (top row of Figure 5g). Meanwhile, CLSM results confirmed the accumulation of Plt-M@P in organs and tumors.
As shown in Figure 5g (bottom row), NPs barely accumulated in the heart and kidney, but small amounts were distributed in the liver, spleen, and lung, with the largest accumulation in the tumor. The The liver has the function of detoxification, which will actively take up some foreign substances and remove them. 83  (h) PI staining was performed to detect the apoptosis of 4T1 cells after PDT. The red channel is PI, and the Merge channel is the superposition of PI and bright field. Ex = 633 nm; Em = 680 nm. Scale bar = 100 μm. Data were reported as mean ± SD and analyzed by two-sided Student's t-test. *p < 0.05，***p < 0.001. CLSM, confocal laser scanning microscopy; PDT, photodynamic therapy; n.s., not significant; ROS, reactive oxygen species.    were randomly separated into four groups (n = 4) as follows: PBS, PDT, anti-PD-L1, and PDT+ anti-PD-L1. The process of tumor therapy is illustrated in Figure 7a. In PDT and PDT+ anti-PD-L1 groups, 100 μl of Plt-M@P were injected via the tail vein every 3 days with a total of four times. At 24 h after the injection, mice were exposed to white light (200 mW cm À2 ) for 15 min. In the PDT+ anti-PD-L1 group, the mice were then intravenously administered with PD-L1 antibody. 85,86 On Day 13 of the treatment, the mice were euthanized and the tumors were removed. Photographs of the tumors indicated that the tumors in mice treated with PDT plus anti-PD-L1 were smaller than those treated with other regimens (Figure 7b,c). For largeburden tumors, combination therapy may have better antitumor effect. Watanabe's study showed that radiotherapy with anti-PD-1 checkpoint blockade enhanced (natural killing) NK and CD8 + T celldependent antitumor immunity in large tumors. 84 In addition, there was no significant change in body weight in each group, indicating an absence of severe systemic toxicity of the NPs (Figure 7d). There was no significant difference for using PDT or anti-PD-L1 immunotherapy alone for large tumors. For PDT, the ineffectiveness may arise from the limited tissue penetration depth of light, whereas for anti-PD-L1, the immunosuppressive microenvironment may be a hindrance to large tumor treatment. Therefore, we investigated why PDT plus anti-PD-L1 can achieve better oncologic outcomes. An important principle of tumor immunology is that tumor cells can be eliminated by T cells. 87 The peripheral blood was collected for quantifying the frac- 3.6 | Biosafety assessment of combination therapy with PDT mediated by Plt-M@P and anti-PD-L1 immunotherapy Conventional tumor treatments, including radiotherapy and chemotherapy, often cause serious side effects, such as hypo-leukemia, liver, and kidney function damage, neuralgia, reproductive toxicity, and so forth. 93,94 The major side effects of PDT are photosensitivity reactions, including local skin redness, oozing, and hyperpigmentation. To reduce the occurrence of these adverse reactions, reducing the accumulation of photosensitizer at nontumor sites is an effective means.
The use of platelet membranes to camouflage the photosensitizer can enhance tumor delivery and reduce its accumulation in normal tissues, thus reducing or avoiding the aforementioned side effects of PDT. In addition, vital organs and functions need to be evaluated to assess the safety of Plt-M@P-mediated PDT in combination with anti-PD-L1 immunotherapy. H&E staining at the end of the anti-tumor study revealed no obvious histological damage in the major organs (spleen, liver, lung, heart, and kidney), supporting the biosafety of Plt-M@P-mediated PDT combination with anti-PD-L1 ( Figure S10a).
In addition, the level of aspartate transaminase (AST) and alanine aminotransferase (ALT) for liver function, creatinine (CRE) and blood urea nitrogen (BUN) for renal function, and the creatine kinase (CK) and lactic dehydrogenase (LDH) showed no difference between PBS group and treatment groups ( Figure S10b). These results further suggested that Plt-M@P-mediated PDT and anti-PD-L1 did not cause functional changes in major organs. In addition, it is well established that cancer treatment, such as radiotherapy and chemotherapy, can deplete the ovarian reserve, resulting in infertility or early menopause in young women. [95][96][97] With cancer survivors rapidly growing, there is now a critical need to develop new treatments to address these complications. To explore the reproductive toxicity of Plt-M@P-mediated PDT and anti-PD-L1, H&E staining of ovary was conducted. The representative histological images of ovaries from four groups exhibited some healthy follicles ( Figure S10c, green, primordial follicles; red, primary follicles; yellow, secondary follicles; blue, antral follicles; orange, atretic follicles). The ovarian function is determined by the number of follicles, different levels of follicles were counted. The total number of follicles in the treatment group (PDT, anti-PD-L1, PDT+ anti-PD-L1) did not differ significantly from that of the PBS group ( Figure S10d). In addition, one of the main functions of the ovary is hormone secretion, the concentrations of 17 β-estradiol (E2) and anti-Müllerian hormone (AMH) in serum were measured. As shown in Figure S10e, the differences in serum E2 and AMH levels between treatment and PBS groups showed no significance, indicating the photodynamic-immunotherapy does not damage ovarian function. Taken together, these results suggested that Plt-M@P-mediated PDT and anti-PD-L1 are safe, and with an almost inexistent toxicity risk to other organs that make it a good candidate for tumor therapy.

| CONCLUSION
In this study, a platelet membrane camouflaged novel AIE photosen-

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.